ProjectSummary TranslesionDNAsynthesis(TLS)polymerasesarecriticaltocellsurvivalbyreplacinghighfidelitypolymerases duringroadblocksthatoccurduringDNArepairandreplication.Onedifficultyinelucidatingthemultiplerolesof thesepolymerasesisthatitisimpossibletoidentifywhichpolymeraseisactiveinaspecificsituation.Herewe proposeachemicalbiologyapproachinwhichwecanmeasuretheactivityofDNApolymerasekappa.We havedesignedandsynthesizedN2-benzyl-2?-deoxyguanosineandanalogsthatarehighlyselecttowardpol kappa.Wehavepreviouslyshownthatinvitro,N2-benzyl-GTPreactswithpol?105-foldmoreefficientlythan poleta,iota,beta,nu,anddelta,andincells,theincorporationofN2-4-ethynylbenzyl-dGintotheDNAis dependentonpolkappa.Withthistoolwewillexaminethemultiplerolesofpolkappaintwofollowingspecific aims:(1)DeterminetheroleofpolkappainNER,and(2)determinetherolepolkappaplaysduringS-phase. Inaim1,wewillexaminetheNERactivityofpolkappawithrespecttoDNAdamage,protein-protein interactions,andthelocationoftheactivityinthegenome.Inaim2wewillexaminepolkappaactivityinS- phasewithrespecttobypassingDNAdamageandreplicationofnon-BDNAsequences.Inparticularwewill examinethepolymeraseswitchmechanismsatthereplicationfork,theroleofprotein-proteininteractionsin activationofpolkappaactivity,andthelocationofpolkappaactivityinthegenome.Similartechniqueswillbe employedinthetwoaims.(i)ActivityassayswillbeperformedutilizingN2-4-ethynylbenzyl-dGandClick Chemistrytoattachafluorophore.Theactivitywillbeanalyzedbyfluorescencemicroscopytoexamine nuclear/cytoplasmiclocalizationof4-ethynylbenzyl-dG,whileflowcytometrywillbeusedtoexaminecell-cycle activity.(ii)Thesetwotechniqueswillbecombinedwithmutant-inactive-proteinstodeterminethecritical proteinsandinteractionsinvolvedintheactivity.(iii)iPOND-likeexperimentswillbeperformedtoidentify proteinsassociatedwiththeactivityinanunbiasedmanner.(iv)DNAstrandfiberassayswillbeemployedto distinguishbetweenthepolymeraseswitchmechanismandpost-gaprepairduringS-phase.(v)Next generationsequencingwillbeutilizedtoprobethegenomicidentityoftheactivity.Thisproposalisvery innovativeincreatinganewmethodbywhichscientistswillbeabletoexaminetheactivityofasingleDNA polymeraseinacell. PUBLICHEALTHRELEVANCE.DifferencesinactivityofDNApolymerasehaveamajorimpactontheability ofanindividualtorespondtoDNAdamagingagents.Thismethodologymaybeusedinidentifyingthe susceptibilityofindividualororganstocarcinogensandtheefficacyofDNAdamagingchemotherapeutic agents.